The purpose of this research is to define the steps involved in mitochondrial iron uptake and heme synthesis. In particular, we propose to idenfity and characterize an iron carrier substance isolated from copper deficient hepatic mitochondria and to determine its role, if any, in the normal movement of iron to the mitochondrial surface. In addition, we will compare the mitochondrial uptake of iron and calcium to determine what elements of the uptake process these two metals have in common. If the similarities of these uptake mechanisms can be clarified, then it may be possible to identify specific factors which regulate mitochondrial iron uptake. We have previously shown that an iron carrier substance can be isolated by centrifugation. Its iron is available for heme synthesis. It is excluded by Sepharose 4B. We will characterize this substance further by additional gel chromatography, as well as by manipulation of metal content and pH. The effect of quantity and duration of iron loading of copper deficient animals will be assessed. The material will also be studied by iron exchange chromatography and polyacrylamide gel electrophoresis. The presence of this substance will be sought in normal mitochondria by methods employing ultrafiltration concentration or, if necessary, by immunochemical methods. Calcium uptake by mitochondria, like iron uptake, is energy dependent. Calcium interferes with heme synthesis, but when calcium is added to mitochondrial in the absence of iron, heme synthesis occurs. Thus, calcium and iron may share a common mitochondrial translocation substance. Using the methods available for identification of calcium carrier, we will study the iron-binding effects of this carrier and determine its role in mitochondrial iron uptake and heme synthesis. Calcium uptake by mitochondria is modified by a number of cations such as Mg ions as by phosphate and several inophores. The effect of these substances on mitochondrial iron uptake and heme synthesis will be evaluated as will this effect of heme.